In bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. Here, we demonstrate that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, our data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways.

The organization of the visual system is different in birds and mammals. In both, retinal axons project topographically to the visual targets in the brain; but whereas in birds visual fibers from the entire retina decussate at the optic chiasm, in mammals, a number of axons from the temporal retina diverge at the midline to project ipsilaterally. Gain-of-function experiments in chick raised the hypothesis that the transcription factor Foxd1 specifies retinal temporal identity. However, it remains unknown whether Foxd1 is necessary for this function. In mammals, the crucial role of Foxd1 in the patterning of the optic chiasm region has complicated the interpretation of its cell-autonomous function in the retina. Furthermore, target molecules identified for Foxd1 are different in chicks and mice, leading to question the function of Foxd1 in mammals. Here we show that in the mouse, Foxd1 imprints temporal features in the retina such as axonal ipsilaterality and rostral targeting in collicular areas and that EphA6 is a Foxd1 downstream effector that sends temporal axons to the rostral colliculus. In addition, our data support a model in which the desensitization of EphA6 by ephrinA5 in cis is not necessary for the proper functioning of EphA6. Overall, these results indicate that Foxd1 functions as a conserved determinant of temporal identity but reveal that the downstream effectors, and likely their mechanisms of action, are different in mammals and birds.

Classic studies have proposed that genetically encoded programs and spontaneous activity play complementary but independent roles in the development of neural circuits. Recent evidence, however, suggests that these two mechanisms could interact extensively, with spontaneous activity affecting the expression and function of guidance molecules at early developmental stages. Here, using the developing chick spinal cord and the mouse visual system to ectopically express the inwardly rectifying potassium channel Kir2.1 in individual embryonic neurons, we demonstrate that cell-intrinsic blockade of spontaneous activity in vivo does not affect neuronal identity specification, axon pathfinding, or EphA/ephrinA signaling during the development of topographic maps. However, intrinsic spontaneous activity is critical for axon branching and pruning once axonal growth cones reach their correct topographic position in the target tissues. Our experiments argue for the dissociation of spontaneous activity from hard-wired developmental programs in early phases of neural circuit formation.